U.S. patent number 9,914,135 [Application Number 12/296,952] was granted by the patent office on 2018-03-13 for methods and apparatus for the selection and/or processing of particles, in particular for the selective and/or optimised lysis of cells.
This patent grant is currently assigned to Menarini Silicon Biosystems S.p.A.. The grantee listed for this patent is Melanie Abonnenc, Nicol Manaresi, Gianni Medoro. Invention is credited to Melanie Abonnenc, Nicol Manaresi, Gianni Medoro.
United States Patent |
9,914,135 |
Manaresi , et al. |
March 13, 2018 |
Methods and apparatus for the selection and/or processing of
particles, in particular for the selective and/or optimised lysis
of cells
Abstract
Methods and apparatus for the selection or processing of
particles sensitive to the application of an external stimulus, in
which is produced, by applying the external stimulus, the
rupture/lysis of at least one selected particle or the fusion of
first and second selected particles, by means of the organization
of the particles using a first field of force by selectively
energizing electrodes of an array of selectable electrodes having
dimensions comparable to or smaller than those of the particles,
applying to the electrodes a first configuration of stresses; and
by applying to the electrodes a second configuration of stresses,
so as to create a second field of force, located substantially
close to at least one selected particle to be lysated or to a pair
of first and second particles to be fused and such as to produce
the application of a stimulus suited to produce their lysis or
fusion.
Inventors: |
Manaresi; Nicol (Bologna,
IT), Medoro; Gianni (Casalecchio di Reno,
IT), Abonnenc; Melanie (Valence, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Manaresi; Nicol
Medoro; Gianni
Abonnenc; Melanie |
Bologna
Casalecchio di Reno
Valence |
N/A
N/A
N/A |
IT
IT
FR |
|
|
Assignee: |
Menarini Silicon Biosystems
S.p.A. (Castel Maggiore, IT)
|
Family
ID: |
38491137 |
Appl.
No.: |
12/296,952 |
Filed: |
April 12, 2007 |
PCT
Filed: |
April 12, 2007 |
PCT No.: |
PCT/IB2007/000963 |
371(c)(1),(2),(4) Date: |
May 01, 2009 |
PCT
Pub. No.: |
WO2007/116312 |
PCT
Pub. Date: |
October 18, 2007 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20090220974 A1 |
Sep 3, 2009 |
|
Foreign Application Priority Data
|
|
|
|
|
Apr 12, 2006 [IT] |
|
|
TO2006A0273 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B03C
5/026 (20130101); C12Q 1/6816 (20130101); C12Q
1/686 (20130101); G16B 20/00 (20190201); B03C
5/005 (20130101) |
Current International
Class: |
B03C
5/00 (20060101); B03C 5/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2002536167 |
|
Oct 2002 |
|
JP |
|
2004503775 |
|
Feb 2004 |
|
JP |
|
WO00/047322 |
|
Aug 2000 |
|
WO |
|
WO00/69565 |
|
Nov 2000 |
|
WO |
|
WO-01096857 |
|
Dec 2001 |
|
WO |
|
WO03/001193 |
|
Jan 2003 |
|
WO |
|
WO03/014739 |
|
Feb 2003 |
|
WO |
|
WO04/071668 |
|
Aug 2004 |
|
WO |
|
WO-2005/075656 |
|
Aug 2005 |
|
WO |
|
WO2007/049103 |
|
May 2007 |
|
WO |
|
WO-2007079663 |
|
Jul 2007 |
|
WO |
|
Other References
Cordero et al, Microelectronic J., vol. 34, pp. 1137-1142 (2003).
cited by examiner .
Lu et al, Lab Chip, vol. 5, pp. 23-29 (2005). cited by examiner
.
International Search Report for PCT/IB2007/000963 dated Sep. 28,
2007. cited by applicant .
Office Action of JP2009-504847 dated Aug. 21, 2012 With an English
Translation. cited by applicant .
Office Action of JP2009-504849 dated Aug. 21, 2012 With an English
Translation. cited by applicant .
K. Maswiwat, et al. Electrochimica Acta. 2006. vol. 51: pp.
5215-5220. Published online May 3, 2006. cited by applicant .
Hughes. Phys. Med. Biol., 1998, vol. 43, pp. 363-3648. cited by
applicant .
Lin et al., Lab Chip. 2004, vol. 4, pp. 104-108. cited by
applicant.
|
Primary Examiner: Crow; Robert T.
Attorney, Agent or Firm: Marshall, Gerstein & Borun
LLP
Claims
The invention claimed is:
1. A method for selecting and processing of first particles
sensitive to the application of an external stimulus and lysing at
least one selected first particle, said first particles being
suspended in a fluid, comprising the steps of: a) providing an
array of electrodes, each electrode having dimensions comparable to
or smaller than those of said first particles and each electrode
being a selectable electrode; b) selectively energizing said array
of electrodes to apply to all of said electrodes a first pattern of
voltages to generate a first field of force having first points of
stable equilibrium each positioned at a first position, wherein the
first particles are attracted towards the first points of stable
equilibrium and are situated at the first positions; c) applying to
all of said electrodes of said array a second pattern of voltages,
different from the first pattern of voltages, to generate a second
field of force having at least one second point of stable
equilibrium positioned at a second position in a gap between at
least two adjacent electrodes in proximity of the at least one
selected first particle to be lysed; wherein the at least one
selected first particle to be lysed is attracted to the at least
one second point of stable equilibrium and shifted to the second
position, wherein: i) said second field of force applies, only at
the second position at which at least one second point of stable
equilibrium is generated, a stimulus to lyse the at least one
selected first particle to be lysed and said second field of force
being designed such that at least one first particle that is not
selected to be lysed remains at the first position; ii) said first
pattern of voltages and said second pattern of voltages are applied
in a time sequence to the same said electrodes; d) after step c)
applying again said first pattern of voltages to said electrodes;
and e) while step d) is in progress, producing a controlled shift
of said fluid to recover a selected product of lysis of said at
least one selected first particle.
2. The method according to claim 1, wherein said stimulus consists
of a localised heating of a fluid in which said at least one
selected first particle to be lysed is immersed.
3. The method according to claim 2, wherein said second pattern of
voltages is such as to produce the selective heating by Joule
effect at the second position at which said second stable point of
stable equilibrium is generated.
4. The method according to claim 2, wherein said second pattern of
voltages is generated in either AC or DC.
5. The method according to claim 1, wherein said first pattern of
voltages for generating said first field of force presents a first
amplitude and a first frequency; and in that said second pattern of
voltages for generating the second field of force presents a second
amplitude and a second frequency, at least one of which is
different from said first amplitude and first frequency.
6. The method according to claim 5, wherein said stimulus consists
of a force applicable to said at least one selected first particle
to be lysed by said second field of force; said second pattern of
voltages being generated in AC.
7. The method according to claim 6, wherein said at least one
selected first particle to be lysed is a biological entity with a
lysable membrane in which said stimulus consists of bringing the
transmembrane potential of said at least one selected first
particle to a value such as to produce the rupture of the
membrane.
8. The method according to claim 1, further comprising checking the
lysis of said at least one selected first particle to be lysed.
9. The method according to claim 1, wherein said first particles
are suspended in a fluid, further comprising choosing the fluid to
present a low electric conductivity compared to an electrical
conductivity of physiological fluids.
10. A method for isolating interesting particles from a population
of particles including said interesting particles and
non-interesting particles, the population of particles being
suspended in a fluid, comprising the steps of: a) introducing said
population of particles suspended in a fluid into a microchamber
equipped with an array of electrodes, each electrode having
dimensions identical with or smaller than those of particles of the
population of particles including said interesting particles and
each electrode being selectable; b) selectively energizing said
electrodes of the array of electrodes to apply to all of said
electrodes a first pattern of voltages to generate a first field of
force having first points of stable equilibrium each positioned a
first position, wherein said interesting and non-interesting
particles from the population of particles are attracted towards
the first points of stable equilibrium and are situated at the
first positions; c) applying to all of said electrodes of said
array of electrodes a second pattern of voltages, different from
the first pattern of voltages, to generate a second field of force
having second stable points of equilibrium each positioned at a
second position arranged in a gap between adjacent electrodes of
said array of electrodes in proximity of the non-interesting
particles, wherein the non-interesting particles of said population
of particles are attracted towards the second points of stable
equilibrium and shifted to the second position and the interesting
particles remain at the first positions, wherein i) said second
field of force is applies solely at the second points of stable
equilibrium a stimulus suitable to selectively lyse the
non-interesting particles ii) said first and second pattern of
voltages are applied in a time sequence to the same said first and
second electrodes; and the method further comprising the step of d)
recovering the interesting particles, wherein recovering the
particles comprises: after step c) applying again said first
pattern of voltages to said electrodes; and while the reapplication
of the first pattern of voltages to said electrodes is in progress,
producing a controlled shift of said fluid to recover a selected
product of lysis of the non-interesting particles.
11. The method according to claim 10, wherein said step c) is
applied simultaneously and/or repeatedly to the non-interesting
particles by applying to said first electrodes a plurality of
voltage patterns suited to apply to said non-interesting particles
stimuli of an intensity and/or type such as to produce the
selective lysis of the non-interesting particles.
12. A method for the ultra-purification of interesting particles
from non-interesting particles comprising contaminated particles,
wherein the isolation method according to claim 10 is applied,
where step c) is applied only to said contaminated particles.
13. A method for the analysis of particles, comprising: a)
introducing said particles, suspended in a fluid, into a
microchamber; b) shifting said particles, selectively, each into a
predetermined point of analysis separate from said microchamber,
but hydraulically connected to it; c) applying to said particles
present in said predetermined points of analysis a method according
to claim 1; d) applying a defined analysis protocol on site to the
respective debris of the lysis of said particles.
14. The method according to claim 13, where said defined analysis
protocol is chosen from the group including: polymerase chain
reaction, capillary electrophoresis on chip; or combinations of
polymerase chain reaction and capillary electrophoresis on
chip.
15. A method for producing the fusion of first particles with
second particles, in which the first and second particles are
biological entities with a lysable membrane, and in which the
membrane of said particles is sensitive to the application of an
external stimulus, the first and second particles being suspended
in a fluid comprising: a) providing an array of electrodes, each
electrode having dimensions identical with or smaller than those of
said first and second particles and each electrode being
selectable; b) selectively energizing said electrodes of said array
of electrodes to apply to all of said electrodes a first pattern of
voltages to generate a first field force having first points of
stable equilibrium, wherein the said first and second particles are
attracted towards first points of stable equilibrium and situated
at a first position in proximity of said first and second
electrodes; and c) applying to all said electrodes of said array of
electrodes a second pattern of voltages, different from the first
pattern to generate a second field of force having at least one
second point of stable equilibrium positioned at a second position
in a gap between at least two adjacent electrodes of said array of
electrodes in proximity to selected first and second particles;
wherein the selected first and second particles are attracted to
the at least one second point of stable equilibrium and shifted to
the second position, wherein i) said second field of force applies
solely at the at least one second stable point of equilibrium a
stimulus suitable to fuse membranes of the selected first and
second particles to be fused together, ii) said first and second
patterns of voltages are applied in a time sequence to the same
said first and second electrodes; and d) after step c) applying
again said first pattern of voltages to said electrodes; and e)
while step d) is in progress, producing a controlled shift of said
fluid to recover the fused first and second particles.
16. The method according to claim 7, wherein said biological entity
is a cell.
17. The method of claim 8, wherein said step of checking the lysis
is carried out by a device with sensors integrated with said array
of electrodes in a single chip.
18. A method for selection and processing of particles sensitive to
an application of an external stimulus, comprising: applying a
first pattern of voltages to an array of electrodes to generate a
first field of force having first points of stable equilibrium,
wherein the particles are attracted towards the first points of
stable equilibrium and reside at a first position and the particles
comprise first particles to be ruptured or lysed and second
particles; applying a second pattern of voltages to the array of
electrodes to generate a second field of force having second points
of stable equilibrium each positioned at a second position between
two adjacent electrodes, wherein the two adjacent electrodes are
disposed in proximity to the first particles such that only the
first particles are attracted to the second points of stable
equilibrium and shifted the second position while the second
particles remain at the first positions, wherein the second field
of force generated by the second pattern of voltages is configured
to also apply electric impulses between the two adjacent electrodes
to apply a stimulus to the first particles in the second position
suitable to rupture or lyse the first particles, wherein the first
and second patterns of voltages are applied in a time sequence to
the same electrodes; applying again said first pattern of voltages
to said electrodes; and while the first pattern of voltages is
being reapplied, producing a controlled shift of said fluid to
recover a selected product of rupture or lysis of said at least one
selected first particle.
Description
TECHNICAL FIELD
The present invention concerns methods and apparatus for the
selection and/or the processing of particles, in particular
particles composed of cells or including cells and/or cellular
material, for example for the selective and/or optimised lysis of
cells, and is applied principally in the implementation of
protocols with resolution on a single cell. The term "processing"
of cells here and below means any type of operation that can be
carried out on a single particle or cell, or on a group of
them.
STATE OF THE ART
The patent PCT/WO 00/69565 to G. Medoro describes an apparatus and
a method for the manipulation and identification/recognition of
particles making use of closed cages with dielectrophoretic
potential and possible integrated sensors. The method described
teaches how to control the position of each particle independently
of all the others in a two-dimensional space. The force used to
trap the particles suspended in a fluid medium is negative
dielectrophoresis. The individual control of the manipulating
operations is achieved by the programming of memory elements and
circuits associated with each element of an array of electrodes and
sensors integrated in the same substratum. The device allows cells
to be isolated, but requires that they be moved towards a second
microchamber, fluidly isolated from the first one. Moreover, no
method is contemplated for transforming the cells.
The U.S. Pat. No. 6,294,063 to Becker et al. describes a method and
apparatus for the manipulation of packages of solid, liquid or
gaseous biological material by means of a distribution of
programmable forces. The patent also mentions the use of sensors.
In this case too the isolation of the cells may take place only by
moving the cells physically through the whole device.
A further force for the manipulation of particles is the force of
viscous friction generated by electro-hydrodynamic flows (EHD),
such as electrothermal flows (ETF) or AC electro-osmosis. In N G.
Green, A. Ramos and H. Morgan, J. Phys. D: Appl. Phys. 33 (2000)
the EHD are used to shift particles. For example PCT WO 2004/071668
A1 describes an apparatus for concentrating particles on
electrodes, exploiting the above-mentioned electro-hydrodynamic
flows.
The Italian patent application BO2005A000481, Medoro et al., lists
some methods for manipulating particles with arrays of electrodes,
and some methods and apparatus for identifying them.
Instead the international patent application PCT/IT02/00524
describes a method in which first biological entities may be
transformed by being put in contact with second biological entities
(for example liposomes containing DNA, or microbeads), where the
first biological entities are immobilised on a surface defined by a
matrix of first electrodes which may be at least in part
selectively activated and directed, placed facing at least one
second electrode, and are contacted with the second biological
entities shifted by means of dielectrophoresis cages.
The patent application PCT IB 2006000636 in the name of the same
Applicant concerns a method and apparatus for the characterisation
and/or count of particles by means of non uniform, time variable
fields of force and integrated optical or impedenziometric sensors.
The fields of force may have positive or negative
dielectrophoresis, electrophoresis or electro-hydrodynamic
movements, characterised by a set of stable points of equilibrium
for the particles (solid, liquid or gaseous); the same method is
suitable for the manipulation of drops (liquid particles)
exploiting known effects such as Electrowetting on dielectric, with
the aim of acting on the control of the position of each particle
present in the specimen, so as to shift said particles in a
deterministic or statistical way, to detect their presence with the
integrated optical or impedenziometric sensors, and/or to
characterise their type, in order to count them or manipulate them
in an efficient way.
In the Italian application in the name of the same Applicant, no.
TO2006A000226 of 27.3.2006, methods and apparatus are described for
the processing (for example washing, incubation, etc.) of particles
in which the particles suspended in a first fluid are introduced
under laminar flow conditions into at least one first microchamber
or first region of the same, in which a second fluid is introduced
under laminar flow conditions into at least one second region of
the microchamber or of a second microchamber, in such a way as not
to mix with the first fluid, and in which at least one field of
force (F) acting on the particles is activated in the
microchamber(s), to provoke a shift of the particles alone in a
predetermined direction and to transfer the same in suspension into
the second fluid; an apparatus' is preferably used including at
least three microchambers arranged in sequence with each other in
one direction and each connected with the microchamber immediately
before it and after it with two orifices offset from each other in
a direction perpendicular to the direction of sequence of the
microchambers.
Recently, in the article A single cell electroporation chip, Lab on
a Chip, 2005, 5 (1), 38-43, Michelle Khine, Adrian Lau, Cristian
Ionescu-Zanetti, Jeonggi Seo and Luke P. Lee, it has been described
how to increase the permeability of the cellular membranes by
electroporation carried out on single cells; in this way, polar
substances which could not otherwise permeate the plasmatic
membrane (such as dyes, medicines, DNA, proteins, peptides and
amino acids) can be introduced into the cell.
The article Flow-through micro-electroporation chip for high
efficiency single-cell genetic manipulation, Sensors and Actuators
A: Physical. Volume 104, Issue 3, 15 May 2003, Pages 205-212, Yong
Huang, Boris Rubinsky describes in particular the genetic
manipulation of individual cells, which is of great interest in
fields such as biology and biotechnologies, obtained by means of an
electroporation chip which makes use of micro-fluid channels to
manipulate single cells with precision; as is known,
electroporation is a technique that uses intense electrical fields
to induce structural re-arrangements in the cell membrane; pores
are thus formed through the membrane when the transmembrane
potential exceeds the dielectric perforation voltage of the
membrane (0.2-1.5V) allowing external substances to penetrate the
membrane and reach the cytoplasm that it contains.
The electroporation of single cells is an interesting technique
also because it allows the study of the variations that occur in a
cell population cell by cell, and also the study of the
intracellular chemistry, for example supplying specific phenotypes
to individual cells by activating or blocking the expression of
specific and individual proteins. Using technology based on the use
of matrices implemented on chips it is therefore possible to
produce apparatus for HTS testing (high throughput screening),
linked both to the expression of DNA and proteins and to chemical
compounds (for example medicines) which are directed towards
specific cell targets (for example receptors).
The electroporation of single cells is also an advantageous
technique in comparison with the protocols of electroporation in
bulk that are normally used, which require very high voltages
(>103 V) and do not allow an efficacious control of the
permeability of the individual cells, so that, for example, it is
difficult to reclose the pores opened previously.
The attempts made so far to achieve the electroporation of single
cells range from the use of carbon fibre microelectrodes (Lundqvist
et al., 1998) to other techniques such as capillaries filled with
electrolyte, micropipettes, and micromanufactured chips.
Micromanufactured devices are ideal both for isolating single cells
and for focussing the electric field.
Lastly, the article "Controlling cell destruction using
dielectrophoretic forces", A. Menachery and R. Pethig, IEE
Proc.-Nanobiotechnol., Vol. 152, No. 4, August 2005, reports on a
study of the lysis of cells for different types of cells in
castellated or polynomial electrodes, and proposes the differential
lysis of cells of different types present in a mixture (choosing
frequencies and amplitudes such as to lysate one type, but save
another).
However, as the electrodes are much bigger than the cells, it is
not proposed to use this approach, and probably it is not possible
to use it, to destroy single cells selectively, irrespective of
their type. In fact, since the position with respect to relatively
large electrodes (and consequently the intensity of the field to
which they are subjected) varies considerably, this method cannot
work homogeneously on the various cells.
Lysis is preferably induced using fields in an interval of
frequencies between the cross-over frequency (beyond which the
cells pass from negative dielectrophoresis (nDEP) to positive
(pDEP)), and lower than the frequency beyond which the potential of
the membrane is attenuated due to the exceeding of the membrane
relaxation constant.
SUMMARY OF THE INVENTION
The aim of the present invention is to supply methods for operating
on fluid samples containing particles, typically cells, for
carrying out the purification and/or isolation of single cells
and/or the transformation of one or more cells, which is without
the limitations and/or the inconveniences described for the prior
art.
In particular an aim of the present invention is to act on the
control of the position of each particle present in the sample, in
order to shift said particles in a deterministic way, to operate
selectively on each cell and/or perform in a more efficacious way
operations such as lysis, fusion, etcetera.
Here and below, the terms "particles" or "particle" are used to
indicate micrometric or nanometric entities, natural or artificial,
such as cells, subcellular components, viruses, liposomes,
niosomes. The term cell will sometimes be used, but where not
specified otherwise it must be understood as a non limiting example
of the use of particles in the sense described more fully
above.
The present invention therefore concerns the methods as specified
in the claims 1, 11, 12, 13, 15.
The invention also concerns an apparatus as specified in claim
17.
In particular, non uniform, time variable fields of force are used
and integrated optical sensors. The fields of force may have
positive or negative dielectrophoresis, electrophoresis or
electro-hydrodynamic movements, characterised by a set of stable
points of equilibrium for the particles.
In this way the limitations of the prior art are overcome by the
present invention.
The implementation of the methods according to the invention allows
the accurate purification of a sample of cells even from
contaminating agents present in a low percentage. It also allows
cells to be transformed efficaciously and selectively with the
introduction of genetic material. Lastly it allows a few
interesting cells to be rapidly isolated from a heterogeneous
sample.
Further characteristics and advantages of the invention will be
clear from the following description of some of its non limiting
embodiments, with reference to the figures in the attached
drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 schematically illustrates the steps of a first method
according to the invention carried out in a manipulation apparatus
illustrated in section in elevation;
FIG. 2 illustrates the steps of the method in FIG. 1 carried out
with the same apparatus as in FIG. 1, but illustrated in layout
viewed from above;
FIG. 3 illustrates in the same view as FIG. 2 a possible variation
of the method in FIGS. 1 and 2;
FIG. 4 illustrates the actuation of the method in FIG. 1 with a
photographic sequence;
FIG. 5 illustrates in the same view as FIG. 2 a further variation
of the method in FIG. 1;
FIG. 6 illustrates in the same view as FIG. 2 a further method of
the manipulation of particles according to the present invention;
and
FIGS. 7 to 9 illustrate different embodiments of an apparatus for a
particularly advantageous actuation of the methods of the
invention.
DETAILED DESCRIPTION
The aim of the present invention is to carry out methods and
apparatus for the manipulation and/or separation and/or analysis of
particles.
The methods of the invention are based (FIG. 1) on the use of a non
uniform field of force (F) with which to attract single particles
or groups of particles (CELL) towards positions of stable
equilibrium (CAGE). This field may be, for example, a field of
dielectrophoresis (DEP), negative (NDEP) or positive (PDEDP), or a
field of electrohydrodynamic movements (EHD).
The processing carried out on the cells is based on the application
of localised electric fields able to provoke the permanent rupture
of the cellular membrane, or the fusion of two particles.
The method may also make use of integrated sensors, preferably of
the optical and/or impedenziometric type, for example in all those
steps in which it is necessary to check the type of particles close
to certain electrodes. Alternatively, similar information may be
available by means of non integrated optical sensors, coupled to a
microscope, which allows the examination of the contents of the
microchamber in which the methods of the invention are being
carried out.
Generation of Forces
There are various methods for generating forces to shift particles,
according to the prior art, by means of arrays of electrodes (EL),
formed on a substratum. Typically, according to previous patents of
the same Applicant (FIG. 1), a cover (LID) is used, which may in
turn be an electrode, which delimits a microchamber, in which are
the particles (CELL), typically suspended in a fluid composed of a
liquid. In the case of dielectrophoresis (DEP), the voltages
applied are periodic in-phase voltages (Vphip) indicated with the
plus sign (+) and counterphase voltages (Vphin) indicated with the
minus sign (-). The term "counterphase voltages" means voltages
offset by 180.degree.. The field generates a force which acts on
the particles of a region of space (CAGE), attracting them towards
a point of equilibrium (PEQ). In the case of negative DEP (NDEP),
it is possible to produce closed force cages, according to the
prior art, if the cover (LID) is a conductive electrode; in this
case the point of equilibrium (MPEQ) corresponds to each electrode
connected to Vphin (-) if the adjacent electrodes are connected to
the opposite phase Vphip (+) and if the cover (LID) is connected to
the phase Vphin (-). This point of equilibrium (MPEQ) is normally
at a distance in the liquid with respect to the electrodes, so the
particles (CELL) are in levitation, in a stationary state.
In the case of positive DEP (PDEP) the point of equilibrium (ZPEQ)
is normally at the surface on which the electrodes are realised,
and the particles (CELL) are in contact with it, in a stationary
state. For the PDEP it is not necessary to have further electrodes
in the cover, because the points of equilibrium of the PDEP
correspond to the maximums of the electric field. For the
electro-hydrodynamic movements (EHD), the configurations of
electrodes generate flows which push the particles towards the
minimum points of the flow.
For the sake of simplicity, below is considered purely as an
example, and therefore without limitation for the purposes of the
present invention, the use of closed cages with negative
dielectrophoresis as the activating force for the steps of particle
movement in the description of the methods and apparatus (for which
it is necessary to use a cover acting as an electrode) of the
invention. To experts of the sector with ordinary abilities it is
clear that it is possible to generalise the methods and apparatus
described below for the use of different activating forces, and
different types of particles.
Method for the Selective Lysis of Particles
The particles to be lysated are positioned close to the gap between
two electrodes by one of the above mentioned actuation forces,
energising the electrodes with sinusoidal voltages of a first
amplitude (MA) and frequency (MF). The gap is preferably smaller
than 10 .mu.m, and typically around 1-3 .mu.m, so that a low
voltage stimulus, compatible with the supply voltage of an
integrated circuit (e.g. 2.5, 3.3 or 5 V), is enough to determine a
transmembrane potential sufficient to cause the irreversible
rupture of the particle.
This stimulus is preferably composed of a train of sinusoidal
impulses of a second amplitude (ZA) and a second frequency
(ZF).
Electric impulses are applied between the two selected electrodes
so as to provoke the lysis of the cell.
FIG. 1 shows in section the evolution over time of the fields of
force and of the "patterns" (that is the complex of configurations
of (+) or (-) state of the electrodes) of the voltages applied to
the electrodes, according to a preferential embodiment of the
invention. In FIG. 1(a), the cells (CELL) are in nDEP, suspended in
the liquid in a first point of equilibrium (MPEQ). In FIG. 1(b) the
pattern of voltages applied to the electrodes (EL) changes, so the
frequency and optionally the amplitude of the voltages applied, as
well as the force to which the cells are subjected, change to pDEP
(FZAP). However, thanks to the change of the pattern of voltages,
only the cell to be lysated (CELLZ) is subjected to a significant
force, so it is attracted towards a new point of stable equilibrium
(ZPEQ). Near that point the electric field is maximum and the
frequency is such that a sufficient transmembrane potential to
lysate the cell is provoked.
FIG. 2 shows in layout the configurations of electrodes for the
same steps (a)-(d) as FIG. 1, where the pattern of electrodes in
phase and counterphase with the voltage applied to the lid is
indicated by the colour (in phase grey, in counterphase white).
This sequence of patterns is particularly favourable since, during
lysis, the other cells in neighbouring areas are subjected to an
almost null electric field, as both the cover and the electrodes
are in phase. If the amplitude of the voltage applied to the cover
is equal to the amplitude of the voltage applied to the electrodes,
the field on those cells is null.
Alternatively, the series of patterns shown in FIG. 3(a)-3(d) may
be adopted. In this case, as is shown by the number of electrodes
in phase and counterphase represented by the colours white and
grey, the advantage lies in the need to reprogramme a smaller
number of electrodes each time, which may be advantageous if the
process of writing the memory cells for the pattern of electrodes
to be actuated is slow. Otherwise it is generally preferable to
adopt the previous solution in FIG. 2.
With sensors integrated in the array of electrodes that delimit the
bottom surface of the microchamber, for example of optical type, it
is easy to check when lysis has occurred, using the methods
described in the above-mentioned international patent application
no. PCT IB 2006000636 of the same Applicant, to see whether the
cage corresponding to the lysated cell is still full or empty, or
better to check for the presence of debris resulting from
lysis.
Substantially, with the methods described it is possible to select
or process particles sensitive to the application of an external
stimulus using a method comprising in general the step of
producing, by applying said external stimulus, the rupture or lysis
of at least, one selected particle; and wherein are also
contemplated the steps of:
a) bringing the particles (CELL) close to electrodes (EL) of an
array of selectable electrodes having dimensions comparable to or
smaller than those of said particles, to which may be applied a
first pattern (PMAN) of tensions to organise optionally, if
necessary, said particles (CELL) by means of a first field of force
(FMAN), by selectively energising said electrodes (EL); b) applying
to the electrodes a second pattern (PZAP) of voltages, so as to
create a second field of force (FZAP), located substantially close
to at least one selected particle to be lysated (CELL) and such as
to produce the application to said at least one selected particle
of a stimulus suited to produce its rupture or lysis.
The particles are suspended in a chosen fluid, in case one wants to
use the passage from nDEP to pDEP as described previously to obtain
lysis, so as to present a relatively low electric conductivity.
The first pattern (PMAN) of voltages for generating the first field
of force (FMAN) presents a first amplitude (MA) and a first
frequency (MF); and the second pattern (PZAP) of voltages for
generating the second field of force (FZAP) presents a second
amplitude (ZA) and a second frequency (ZF), at least one of which
is different from said first amplitude (MA) and first frequency
(MF). In this case, the stimulus applied to obtain lysis consists
of a force that can be applied to the at least one selected
particle by the second field of force (FZAP) and both the first and
the second pattern of voltages are generated in AC (alternating
current). In particular, the at least one selected particle is a
biological entity with a lysable membrane, in the examples
described a cell, and the stimulus applied consists of bringing the
transmembrane potential of the at least one selected particle to a
value such as to produce the rupture of the membrane.
According to a possible variation of the method of the invention,
which may be considered illustrated in FIG. 2(b), the stimulus
applied to the selected particle to lysate it consists vice versa
of heating located in the fluid in which is suspended the selected
particle to be lysated.
According to this possible variation, particularly advantageous if
the particles are suspended in a fluid (liquid) presenting a high
electric conductivity (for example physiological solution), the
second pattern (PZAP) of voltages is such as to produce the
selective heating by Joule effect of those selected electrodes in
the array of electrodes with which the second field of force (FZAP)
is generated, in FIG. 2(b) the electrode shown in white, on which
the whole current supplied to the device illustrated is practically
concentrated.
In this case it is clear that at least the second pattern of
voltages may be generated in either AC or DC (direct current).
Anyway, the methods described according to the invention include a
step of checking the lysis of the at least one selected particle,
preferably carried out by means of the already mentioned sensors
integrated with the array of electrodes, in a single chip.
Lastly, according to a further possible variation of the methods
described, if one is interested in selectively recovering the
debris produced by lysis, after the step b) described above, the
following steps may be carried out:
c) applying said first pattern (PMAN) of voltages to the electrodes
again; and
d) while step c) is in progress, producing a slow and controlled
shift of the fluid to recover a selected product of lysis of the at
least one selected particle.
In fact, as is well known to experts in the field, in the case of
actuating the movement of the particles by dielectrophoresis, the
forces acting on the particles due to the applied field are in
proportion to the cube of the radius of the particles, while the
forces of hydrodynamic viscous friction are in proportion only to
the radius of the particles; therefore the smallest particles (the
debris of lysis in this specific case) may be carried along by a
moderate flushing of the fluid in which the particles are
suspended, while the largest particles (the non lysated cells) are
kept in a stationary position (against the viscous flushing action)
by the nDEP cages positioned in stationary mode and in which the
cells are trapped.
The efficacy of the methods described, in particular of the method
according to the FIGS. 1 and 2, is shown in FIG. 4. Two Raji cells
suspended in an aqueous solution with Mannitol 280 mM (millimolar)
and KCl 6.25 mM are organised, see FIG. 4(a), by the electric field
(MF) applied (MA) in which the electrodes in the array have
sinusoids with a peak-peak amplitude of 3.3 V, the conductive cover
(LID) an amplitude of 6.6V, all with frequency 50, kHz. The cells
are taken onto the in-phase electrodes with the lid surrounded by
electrodes of the opposite phase (offset by 180.degree.).
After that, the pattern of the voltages applied to the electrodes
varies, putting into counterphase also the electrode on the cell at
bottom right, which must be preserved. Although there is no cage,
the cell remains in the same position, due to inertia.
Instantaneously, the applied electric field changes so as to
produce positive dielectrophoresis (FZAP), bringing the frequency
of the electric field (ZF) to 400 kHz, and the particle still
present in the cage (CELLZ) goes into a new point of stable
equilibrium due to the force of positive dielectrophoresis, now
generated by the field, which is on the gap between the electrodes,
see FIG. 4(b). That region corresponds to the maximums of the
electric field, which at that frequency are sufficient to provoke
the lysis of the membrane, see FIG. 4(c).
It appears clear to experts in the sector with ordinary abilities
that the electric field may be varied, in different ways, in
particular also (or only) in amplitude, or the initial pattern of
electrodes for manipulation and lysis may be chosen differently,
for example as in FIG. 5.
In this case, it starts from a nDEP pattern, FIG. 5(a), which
positions the particles in a point of equilibrium (MPEQ) lying
vertically to the point of equilibrium for the pDEP (ZPEQ), for the
next pattern of electrodes. In this way the selected cell does not
move from its vertical line, and the lysis process can be
accelerated because the cell takes less time to reach the area of
maximum electric field in which lysis takes place.
Method for Particle Fusion Assisted by Dielectrophoretic
Manipulation
In this case, two particles are brought into contact in the same
point of stable equilibrium (MPEQ) by the force (F) generated by
the electrodes (EL). The stimulus that is applied next to the pair
of cells in contact is chosen with an amplitude and frequency such
as to provoke a fusion of the two cellular membranes into a single
entity (FIG. 6).
With sensors integrated in the chip that holds the array of
electrodes, for example of an optical type, it is then possible to
check that fusion has taken place without the need of an external
microscope.
Applications of fusion include for example the generation of
hybrids, of both eucariot cells and bacteria, or of plants.
For example, one could consider the possibility of using a similar
method to reprogramme differentiated cells towards stem cells, for
example combining a differentiated cell with an enucleated stem
cell.
So, according to this method, the fusion of first particles with
second particles is produced, in which the first and second
particles are biological entities with a lysable membrane, for
example cells or micro-organisms, and in which the membrane of the
particles is sensitive to the application of an external stimulus,
performing the steps of:
a) bringing said first and second particles (CELL) close to
electrodes (EL) of an array of selectable electrodes having
dimensions comparable to or smaller than those of said particles,
to which may be applied a first pattern (PMAN) of tensions to
organise optionally, if necessary, said first and second particles
(CELL) by means of a first field of force (FMAN), by selectively
energising said electrodes (EL); b) applying to the electrodes a
second pattern (PZAP) of voltages, so as to create a second field
of force (FZAP), located substantially close to at least one first
and one second selected particles to be fused together and such as
to produce the application to the same of a stimulus suited to
produce the fusion of the membranes of the first and second
selected particles. Method of Isolating Cells by Survival
A multiplicity of cells is flushed in suspension in the
microchamber with the array of electrodes. Using integrated sensors
(for example optical and/or impedenziometric) and/or external
sensors (for example an optical sensor coupled to a microscope,
with or without fluorescence), the type or cell found in each point
of equilibrium (PEQ) is identified. Stimuli for lysis are then
applied selectively to all the cells that are not interesting,
preserving the vitality of the neighbouring interesting cells.
The sample is then flushed out, recovering the interesting live
cells and the lysate of non interesting cells.
According to the description, a method is therefore carried out for
isolating interesting particles from a population of particles
including the interesting particles, characterised in that it
comprises the following steps:
a) introducing the population of particles, suspended in a fluid,
into a microchamber, where the microchamber is provided with an
array of selectable electrodes;
b) applying to said population of particles the method of selective
lysis described above to produce the selective lysis of all the
particles of the population except the interesting ones;
c) recovering the interesting particles.
Step b), particularly in the case where the position of the
interesting particles with respect to the array of electrodes is
not known a priori and the interesting particles possess
characteristics such as to be sensitive only to a determined
intensity and/or type of stimulus (also not known a priori), is
applied simultaneously and/or repeatedly to all the particles of
the particle population by applying to said electrodes a plurality
of voltage patterns suited to apply to said particles stimuli of an
intensity and/or type such as to produce, in this way, the
selective lysis of all the particles in the particle population
except the interesting ones.
The different type may for example include the application of AC
voltages with determined frequencies, known to be ineffective in
the lysis of the interesting particles, but to be efficacious for
the lysis of the remaining particles. The different intensity may
for example be a growing intensity.
With respect to isolation based on moving the interesting cells
into a second microchamber for recovery, the method described above
presents the following advantages:
1. Speed of Execution
The cells move slowly under the described forces (DEP, ETF, EHD) of
actuation, and the sorting based on moving the cells into a
microchamber for recovery is therefore relatively slow. Vice versa,
the time to complete the sorting operation based on survival
requires only one cell to have reached the nearest point of stable
equilibrium (PEQ), and the time of lysis of the cell (about one
second), and it is not necessary to move it through the whole
selection microchamber.
2. A Cooling System is not Necessary
The cells to be eliminated can be lysated in series, working by
sub-regions of the microchamber. In this way it is not necessary to
have cooling systems, even for working on very large chips with
relatively conductive buffers, because the quantity of heat
developed is proportional to the energised area, which may be made
as small as one likes.
3. The Chip May have Very Large Dimensions
As it is not necessary to energise the whole chip at the same time,
the problem of resistive drop on the tracks that carry the stimuli
to the various electrodes is correspondingly reduced. Above all
with relatively conductive buffers, and with low pitch between
electrodes, the resistive drop on the tracks inside the chip and/or
the drop on the conductive layer of the lid (when NDEP cages are
used for actuation) is not negligible if the whole array of
electrodes is energised. Energising only a part of the layer, the
resistive load to be managed is limited and the voltage drop on the
tracks is decreased.
4. Operation Also with Cells that Cannot be Manipulated with the
Fields of Force
If the electrode matrix is sufficiently dense (with dimensions
comparable to or smaller than the cells), so as to be able to
perform selective lysis on cells even without having selected them
beforehand, the method of the invention can still be completed, in
particular if the cells to be preserved, presenting different
characteristics from the non interesting cells, are sensitive to
stimuli carried out at different frequencies of voltages applied to
the electrodes.
5. Simplified Microfluidic Package
It is not necessary to have a double microchamber, but a single
compartment is sufficient, and the recovery need not be selective,
so the contamination is not linked to the characteristic of the
recovery flow.
Any sensors integrated in the chip will be used to
1. determine the interesting particles.
2. Check the lysis of the undesired cells.
Method of Ultra-Purification of Cells
With enrichment techniques it is often easy to eliminate cells
present in proportions greater than the interesting cells by
several degree of magnitude (for example with centrifugations in
gradients of density or enrichment with magnetic balls, etc.).
However it is sometimes difficult to eliminate the few
contaminating cells that remain (for example present in a
proportion of 0.1-10%) to obtain a 100% pure sample. This is
required for example if one wants to cultivate the interesting
cells but these proliferate less rapidly than the non interesting
ones, which, even if present in a low percentage, would then
prevail downstream from the proliferation.
As in the selection method described previously, the cells are
introduced into the microchamber, the cells optionally line up in
points of stable equilibrium near the electrodes (PEQ), and the
contaminating cells are eliminated by lysating them with the
electrodes, obviously after having identified them with suitable
integrated or external sensors, or on the basis of intrinsic
differences such as the frequency of the voltage applied to the
electrodes which produces lysis.
In this case too, the integrated sensors, if they exist, can
optionally be used to
1. determine the interesting particles.
2. Check the lysis of the undesired cells.
The 100% pure cells are then recovered, flushing out the
sample.
Based on this aspect of the invention, a method is therefore
actuated for the ultra-purification of interesting particles from
contaminating particles, both contained in a population of
particles, characterised in that the isolation method described
above is applied, where step b) is applied only to the
contaminating particles.
Method of Cell Analysis
The study at biomolecular level of DNA and/or of proteins in single
cells is increasingly interesting. According to an aspect of the
present invention, a method is proposed for selecting and shifting
selected cells possibly from a multiplicity of cells from a first
multiplicity of points, and bringing them to a second multiplicity
of points of analysis of micrometric dimensions, each point of
analysis comprising at the most one single cell. The cell is
lysated and its content is analysed on the chip, for example
according to known techniques such as PCR and/or capillary
electrophoresis on chip.
Based on the invention, a method is therefore supplied for the
analysis of particles, characterised in that it comprises the steps
of:
a) introducing said particles, suspended in a fluid, into a
microchamber;
b) shifting said particles, selectively, each into a predetermined
point of analysis separate from said microchamber, but
hydraulically connected to it;
c) applying to said particles present in said predetermined points
of analysis a method of selective lysis as described above;
d) applying a defined analysis protocol on site to the respective
debris of the lysis of said particles; where said defined analysis
protocol is chosen from the group including: PCR, capillary
electrophoresis on chip; combinations of the previous ones.
Cell Analysis Apparatus
To carry out the methods described, in particular the previous
method of analysis, an apparatus is preferentially used as
illustrated in the FIGS. 7, 8 and 9. The apparatus (FIG. 7)
contains an array of electrodes as in the prior art, but it is
characterised by a main microchamber (CHM) and by a multiplicity of
secondary microchambers (CHJ). The main microchamber may be filled
with a sample comprising at least one cell through the respective
inlets (IM1) and outlets (OM1). Each secondary microchamber (CHJ)
is of larger dimensions, preferably substantially comparable to
those of a cell, as shown in FIG. 7. Preferably each secondary
microchamber is connected to the main microchamber through a
channel (LCHG) with a configuration (length and/or shape)
sufficient to prevent (avoid or at least limit) the dispersion of
the sample by diffusion and contamination towards other
microchambers, in the time necessary for the analysis. According to
a possible variation (FIG. 8), there is a multiplicity of secondary
microchambers for lysis (CHLJ) connected to a channel for capillary
electrophoresis on chip, for example with a cross junction (TJ).
Alternatively a series of channels for capillary electrophoresis
may be produced with a double T junction, according to the prior
art. Optionally, at the end of the channel for capillary
electrophoresis there is an integrated sensor (SENS_J), of an
impedenziometric and/or optical type, able to produce an
electropherogram based on the migration time of the compounds
analysed from the junction (cross or double T) to the sensor
itself. A further variation is illustrated in FIG. 9, where each
microchamber of said multiplicity is connected to a capillary for
electrophoresis (CAPJ) through a fluid outlet (OJ) of each
secondary microchamber.
* * * * *